System for optical stimulation of target cells

ABSTRACT

Various systems and methods are implemented for controlling stimulus of a cell. One such method is implemented for optical stimulation of a cell expressing an NpHR ion pump. The method includes the step of providing a sequence of stimuli to the cell. Each stimulus increases the probability of depolarization events occurring in the cell. Light is provided to the cell to activate the expressed NpHR ion pump, thereby decreasing the probability of depolarization events occurring in the cell.

RELATED PATENT DOCUMENTS

This application is a continuation of U.S. patent application Ser. No.15/623,081, filed Jun. 14, 2017, now U.S. Pat. No. 10,105,551, which isa continuation of U.S. patent application Ser. No. 15/229,064, filedAug. 4, 2016, which is a continuation of U.S. patent application Ser.No. 14/886,763, filed Oct. 19, 2015, which is a continuation of U.S.patent application Ser. No. 14/665,978, filed Mar. 23, 2015, now U.S.Pat. No. 9,187,745, which is a continuation of U.S. patent applicationSer. No. 14/219,547, filed Mar. 19, 2014, which is a continuation ofU.S. patent application Ser. No. 13/763,119, filed Feb. 8, 2013, nowU.S. Pat. No. 8,864,805, which is a continuation of U.S. patentapplication Ser. No. 12/522,520, filed Jan. 8, 2010, now U.S. Pat. No.8,398,692, which is a U.S. national phase filing under 35 U.S.C. § 371of PCT/US2008/050745, filed Jan. 10, 2008, which application claimsbenefit under 35 U.S.C. § 119(e) both of U.S. Provisional ApplicationNo. 60/879,669 filed on Jan. 10, 2007 and entitled“Genetically-Targetable Optical Inactivation of Excitable Cells” and ofU.S. Provisional Application No. 60/903,248 filed on Feb. 23, 2007 andentitled “Genetically-Targetable Optical Inactivation of ExcitableCells,” each of which applications is fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates generally to systems and approaches forstimulating target cells, and more particularly, to using optics todissuade stimulation-generated pulse trains.

BACKGROUND

Various efforts in neuroscience are directed towards determining whetherneural activity in a specific brain region, or in a set ofgenetically-identified neurons, contributes to a particular neuralcomputation, behavior, or neurological or psychiatric disorder. Forcenturies, insights have come from studies of human patients withspecific lesions, as exemplified by Paul Broca's delineation in the1860s of the eponymous brain area that, when dysfunctional, results indeficits of speech production. Many studies have used ablation orpharmacological shutdown of neurons or brain regions in animals, orcareful analysis of patients, to parse out the physical substrates ofnormal and abnormal behavior. However, growing awareness that activityin multiple brain regions may be coordinated during performance of abehavior, or in a particular neural dysfunction, has raised the questionof precisely when specific brain regions or neurons contribute. Forexample, a large number of in vivo recording studies have demonstrated,for many brain regions, that specific neurons can fire action potentialsduring precise intervals within a behavioral task. The intervals canlast as little as a fraction of a second; it is possible that specificbrain regions or neurons are required only at specific times in a task,not continuously. In humans, use of transcranial magnetic stimulation todisrupt the visual cortex has demonstrated that conscious perceptionrequires intact cortical performance during temporal windows that lasttens of milliseconds, occurring at precise times after visual stimuluspresentation. Accordingly, a method for disrupting activity in targetedcell types for very precisely delimited periods of time (e.g., severalmilliseconds) could help answer a number of outstanding questions, andenable novel ones to be asked. For example, one question involves theidentification of the precise brain regions, cell types, and activitypatterns required at each phase (sensory, decision-making and motor) ofa behavioral task. Another question involves, for a particularperception (e.g., feeling, decision, memory, or action) identifying theprecise number of neurons that must be active within a certain regionand how long the neurons are active. Another question involves theidentification of the causal role of neural synchrony and precise spiketiming in neural computation, plasticity, and pathological brainfunction. As memories are encoded, consolidated, and forgotten, it canbe important to identifying how the critical neural loci of memorychanges.

SUMMARY

The claimed invention is directed to photosensitive bio-molecularstructures and related methods. The present invention is exemplified ina number of implementations and applications, some of which aresummarized below.

According to one example embodiment of the present invention, a methodis implemented for optical stimulation of a cell expressing an NpHR ionpump. The method includes the step of providing a sequence of stimuli tothe cell. Each stimulus increases the probability of depolarizationevents occurring in the cell. Light is provided to the cell to activatethe expressed NpHR ion pump, thereby decreasing the probability ofdepolarization events occurring in the cell.

The above summary of the present invention is not intended to describeeach illustrated embodiment or every implementation of the presentinvention. The figures and detailed description that follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thedetailed description of various embodiments of the invention thatfollows in connection with the accompanying drawings, in which:

FIG. 1A shows sample outward currents elicited by two pulses of yellowlight in a voltage-clamped neuron, consistent with an embodiment of thepresent invention;

FIG. 1B shows sample membrane voltage hyperpolarizations elicited by twopulses of yellow light, in a current-clamped neuron held at restingmembrane potential, consistent with an embodiment of the presentinvention;

FIG. 1C shows Kinetic properties of yellow light-elicited, Halo-mediatedcurrents from voltage-clamped neurons, consistent with an embodiment ofthe present invention;

FIG. 1D shows membrane potentials of neurons expressing Halo-GFP andexposed to yellow light, neurons expressing Halo-GFP but not exposed toany light, and neurons without transfection with Halo-GFP, consistentwith an embodiment of the present invention;

FIG. 1E shows sample membrane hyperpolarizations induced by 5 Hz and 10Hz trains of yellow light pulses, consistent with an embodiment of thepresent invention;

FIG. 2A shows three voltage traces of a current-clamped hippocampalneuron, exposed to a Poisson train of yellow light pulses, consistentwith an embodiment of the present invention;

FIG. 2B shows voltage traces of three different current-clamped neuronsexposed to the same Poisson train of light pulses (λ=100 ms), consistentwith an embodiment of the present invention;

FIG. 2C shows properties of hyperpolarization events elicited by Poissontrains with various inter-pulse intervals, consistent with an embodimentof the present invention;

FIG. 2D shows a comparison of the peak hyperpolarization and thetime-to-peak data at the beginning and end of the Poisson trains, forthe neurons described in FIG. 2C, consistent with an embodiment of thepresent invention;

FIG. 3A shows a light-driven spike blockade, demonstrated for a singlehippocampal neuron, consistent with an embodiment of the presentinvention;

FIG. 3B shows population data (n=6 neurons) for light-driven,Halo-mediated spike blockade, consistent with an embodiment of thepresent invention;

FIG. 4A shows responses of single neurons co-expressing Halo and ChR2,both under control of the CaMKII promoter, to rapidly-switched pulses ofyellow and blue light, consistent with an embodiment of the presentinvention; and

FIGS. 4B-4D show poisson trains of rapidly-alternating yellow and bluelight pulses elicited rapidly-alternating hyperpolarizations anddepolarizations in the same neuron, consistent with an embodiment of thepresent invention.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION

The present invention is believed to be useful for enabling practicalapplication of a variety of photosensitive bio-molecular structures, andthe invention has been found to be particularly suited for use inarrangements and methods dealing with neuron stimulation. While thepresent invention is not necessarily limited to such applications,various aspects of the invention may be appreciated through a discussionof various examples using this context.

The aspects of the present invention are directed to a technology thatenables rapid neural inactivation and release from inactivation at themillisecond timescale, is safe and effective, has minimal effects oncellular physiology or survival, and requires no exogenous chemicals tobe delivered. A specific embodiment of the invention involves asingle-component protein capable of mediating light-induced inhibition,the mammalian codon-optimized version of the light-driven chloride pumphalorhodopsin, from the archaebacterium Natronobacterium pharaonic(abbreviated Halo). Although such halobacteria are known to live in veryhigh saline concentrations (e.g., >1 M), some wild-type halorhodopsinshave been shown to preserve functionality at much lower chlorideconcentrations, even at levels comparable to those found in mammaliancerebrospinal fluid. Applications of the present invention involve theuse of Halo to mediate optical inhibition of neuronal spiking in aphysiologically accurate milieu, in response to pulses of somaticallyinjected intracellular current (˜300 PA), with temporal onset and offsetof inhibition in the range of 10-15 milliseconds. Moreover, Halo canmediate naturalistic trains of inhibitory voltage changes atphysiologically relevant frequencies, with minimal attenuation ofvoltage amplitude from pulse to pulse.

Aspects of an embodiment of the invention are also directed to a singleneuron expressing both Halo and the blue-light driven cation channelChannelrhodopsin-2 (ChR2), neural inhibition and excitation arecontrolled at the millisecond timescale by pulses of yellow and bluelight, respectively. In one instance, these channels provide thecapability to create lesions of virally or transgenically targetedneural circuits over precise timescales, as well as neuroengineeringinterfaces for bi-directional control of excitable cell depolarizationand hyperpolarization.

One embodiment of the present invention involves a designed fusionprotein having the mammalian codon-optimized form of N. pharaonishalorhodopsin (Halo), with EGFP added in-frame at the C-terminus forease of visualization. When expressed using the CaMKII promoter, whichtargets excitatory neurons of the forebrain, Halo-EGFP fluorescedbrightly and appeared evenly distributed in the neuron. When exposed to˜10 m W/mm² yellow light (e.g., from a xenon lamp, filtered by astandard Texas red excitation filter (bandpass, 560±27.5 nm, Chroma),voltage-clamped hippocampal neurons expressing Halo can experienceoutward currents with rapid onset, stable steady-state, and abruptshut-off with cessation of illumination. In some instances, nosupplementation of the culture medium or the recording medium with thehalorhodopsin cofactor all-trans retinal is necessary. This is believedto be due to levels of all-trans retinal naturally occurring inmammalian neurons in culture and in live brain that are high enough toenable type I opsins without chemical supplementation.

FIG. 1 shows the results of an experimental test ofmillisecond-timescale, yellow light-driven, neuronal hyperpolarizationwith Halo. A cultured hippocampal neuron expressing mammaliancodon-optimized N. pharaonic halorhodopsin (Halo) fused to GFP under theCaMKII promoter is used.

FIG. 1A shows sample outward currents elicited by two 1-second pulses ofyellow (560±27.5 nm) light (˜10 mW/mm²) in a voltage-clamped neuron heldat −70 mV. Yellow bars in this and subsequent figures indicate theperiod of yellow light exposure.

FIG. 1C shows Kinetic properties of yellow light-elicited, Halo-mediatedcurrents from voltage-clamped neurons. FIG. 1Ci shows 15-85% currentonset time. FIG. 1Cii shows 85-15% offset time. For each measurement,data is presented from neurons held at −70 mV (n=14 neurons), −30 mV(n=10), and +10 mV (n=10) (left to right). Bars represent mean±standarderror of the mean (S.E.M.).

FIG. 1B shows sample membrane voltage hyperpolarizations elicited by two1-second pulses of yellow light, in a current-clamped neuron held atresting membrane potential.

FIG. 1D shows membrane potentials of neurons expressing Halo-GFP andexposed to yellow light (left, n=14), expressing Halo-GFP but notexposed to any light (middle, n=11), and without transfection withHalo-GFP (right, n=8). *** denotes significant difference between theHalo-GFP+light condition and each of the other two conditions (p<0.0001;Fisher's partial least-squares difference (PLSD) post hoc test afterANOVA).

FIG. 1E shows sample membrane hyperpolarizations induced by 5 Hz (top)and 10 Hz (bottom) trains of yellow light pulses, with light pulsedurations of 50 ms (top) and 25 ms (bottom), respectively.

In related experimental tests, the light pulses elicited pulseamplitudes of 56.9±23.4 pA (mean±st. dev.; n=14 neurons). Repeating a1-second pulse of yellow light twice, spaced by 1 second in darkness,resulted in identical pulse amplitudes each time (p>0.50, pairedt-test), as shown in FIG. 1A.

This stable current amplitude appears to be consistent with what isknown about the halorhodopsin photocycle. As befits a chloride pump, thecurrent amplitude did not vary significantly with holding voltage(F=0.004, p>0.95, ANOVA with factor of holding voltage), nor did anymeasured kinetic parameters vary, such as the onset or offset times ofthe current pulses (F<0.6, p>0.55 for all comparisons, ANOVA; FIG. 1C).The onset and offset times of elicited currents were seen to be on theorder ˜10-15 ms at all holding voltages tested. This suggests that Halois a viable candidate for ultratransient shutdown of spike trains (FIG.1Ci, 1Cii). When held in current clamp, hippocampal neurons underwentpeak hyperpolarizations of −21.6±11.3 mV (mean±st. dev.; n=11 neurons)in response to pulses of yellow light, with no difference between thepeak hyperpolarizations achieved by two pulses separated by a 1-secondpause (p>0.85, paired t-test; FIG. 1B). These large voltage changes wererelatively rapid, with onset and offset times of 68±57 and 73±39 ms,respectively. Thus, Halo has been shown to be capable of reliablymediating hyperpolarizations of significant magnitude, with fast onsetand offset times at the beginning and end of light exposure.

Several control experiments were implemented to evaluate whether Halohas unanticipated side effects, such as altering basal cell physiologyor increasing the propensity for cell death. First, the basal state ofHalo-expressing neurons electrophysiologically was characterized when nolight was present. When measured in darkness, no difference was seenbetween the resting potentials of neurons expressing Halo and those ofneighboring neurons in the culture that were untransfected (p>0.20, n=11Halo-positive cells, n=8 Halo-negative cells; FIG. 1D). This resultsuggests that basal neural activity would be little affected by thepresence of Halo. On the other hand, Halo-expressing neurons illuminatedwith yellow light were significantly hyperpolarized, with respect toboth Halo-expressing neurons in darkness and non-transfected cells(p<0.0001 for both of these comparisons, Fisher's partial least squaresdifference post hoc test after ANOVA (F=28.4, p<0.0001) with factor ofexperimental condition; FIG. 1D). An independent assay for unanticipatedeffects on cell health, the membrane-impermeant DNA stain ethidiumhomodimer-1 was used to detect the cell membrane breakdown accompanyingcell death for one week in Halo-expressing cells. Little difference wasfound in the prevalence of cell death between Halo-positive andHalo-negative neurons: 16/308 (5.2%) non-transfected neurons counted,and 1/22 (4.5%) Halo-expressing neurons counted, were labeled byethidium homodimer-1, indicating that Halo was not toxic over the courseof the one-week experiment (x2=0.02, p>0.85).

In an effort to explore the uses Halo could present in the analysis andengineering of intact neural circuits, an experiment was performed todetermine whether the fast response times of Halo could supportnaturalistic sequences of hyperpolarization events, in response totrains of brief pulses of yellow light.

FIG. 2 shows high-fidelity Halo-mediated naturalistic trains ofinhibitory events. FIG. 2A shows three voltage traces of acurrent-clamped hippocampal neuron, exposed to a Poisson train of yellowlight pulses. Each light pulse lasts 10 ms, and the Poisson train has amean inter-pulse interval of λ=100 ms.

FIG. 2B shows voltage traces of three different current-clamped neuronsexposed to the same Poisson train of light pulses (λ=100 ms).

FIG. 2C shows properties of hyperpolarization events elicited by Poissontrains with inter-pulse interval λ=100 ms (i, ii) and λ=200 ms (iii,iv), plotted versus onset time of each light pulse. Plots (i) and (iii)show the peak of each hyperpolarization event, as well as theacross-trials standard deviation of these amplitude values across tentrials. Plots (ii) and (iv) show the latency between the onset time ofthe light pulse and the time of the hyperpolarization peak, as well asthe across-trials standard deviation of these timing values across tentrials. All plotted points are across-neuron mean±S.E.M. (n=5 neurons).

FIG. 2D shows a comparison of the peak hyperpolarization (i) and thetime-to-peak (ii) data at the beginning (first 5) and end (last 5) ofthe λ=100 ms and λ=200 ms Poisson trains, for the n=5 neurons describedin FIG. 2C. In (i): for each neuron, the average of the first 5 or last5 hyperpolarization peaks or the across-trials standard deviation ofthese amplitude values was first computed, then the across-neuronmean±S.E.M. was plotted. In (ii): for each neuron, the average of thefirst 5 or last 5 times-to-peak or the across-trials standard deviationof these times-to-peak were first computed, then the across-neuronmean±S.E.M. was plotted.

FIG. 2A shows three traces of hyperpolarization events elicited in asingle neuron, resulting from repeatedly playing back a Poisson train(mean inter-pulse interval, λ=100 ms, 59 pulses), of 10 ms-durationyellow light pulses, to simulate stochastic synaptic inhibitory input.FIG. 2B shows three such hyperpolarization traces, taken from differentneurons. The variability of such trains was remarkably low in manyregards—across ten repeated trials in a single cell, across multiplecells (n=5 neurons), and over time throughout a sustained train of 59pulses (FIG. 2C, 2D). It was found that for hyperpolarizations elicitedby 10 ms-duration light pulses during a λ=100 ms Poisson train, the meanamplitude was −4.56 mV (averaged across trials and neurons), but thetrial-to-trial standard deviation of this amplitude was only 0.40 mV(averaged across neurons, FIG. 2Ci and FIG. 2Di). The trial-to-trialjitter of the time the hyperpolarization took to reach its peak valuewas also small, 1.27 ms (averaged across neurons, FIG. 2Cii and FIG.2Dii). The neuron-to-neuron variability of amplitude and timing wassomewhat larger than the trial-to-trial variability, with standarddeviations of 1.45 mV and 1.78 ms, respectively, but demonstrating thatprecise inhibitory control of a population of neurons could proceed withmillivolt and millisecond resolution. Finally, the through-trainsustainability of light-elicited voltage changes was quantitativelyexamined by comparing the amplitude mean and amplitude variability, andtiming variability of the hyperpolarization events elicited by the firstfive light pulses to those of the last five light pulses in the train(FIGS. 2Di and 2Dii, left side). Little or no difference was seen forany of these statistics between the beginning and end of a train (p>0.10for all measures, t-test). Identical conclusions held for the λ=200 msPoisson train with 46 pulses (FIGS. 2Ciii and 2Civ, and FIGS. 2Di and2Dii, right side). The high temporal and amplitude fidelity ofHalo-mediated hyperpolarizations suggests uses for Halo in simulatinginhibitory synaptic inputs, with great precision.

FIG. 3 shows reliable and repeatable Halo-mediated neural inactivation,at single-spike temporal resolution. FIG. 3A shows a light-driven spikeblockade, demonstrated for a single hippocampal neuron. At the top ofFIG. 3A, labeled with “I-injection,” neuronal firing of 20 spikes at 5Hz are induced by pulsed somatic current injection (˜300 pA, 4 ms). Inthe middle of FIG. 3A, labeled with “light,” light membranehyperpolarizations are induced by two periods of yellow light, timed soas to be capable of blocking spikes 7-11 and spike 17 out of the trainof 20 spikes. At the bottom of FIG. 3A, labeled as “I-injection+Light”,yellow light drives Halo to block neuron spiking (note significantreductions of spikes 7-11 and of spike 17), while leaving spikeselicited during periods of darkness largely intact.

FIG. 3B shows population data (n=6 neurons) for light-driven,Halo-mediated spike blockade, showing high spike probability duringperiods of darkness (spikes 1-6, 12-16, and 18-20), and low spikeprobability during periods of yellow-light illumination (spikes 7-11 andspike 17). Error bars are smaller than the points plotted.

Such experiment were implemented to analyze the ability of Halo toenable rapidly inducible and reversible silencing of neuron spiking.Such ability can be useful to enable time-resolved parsing of theprecise neural substrates of behavior. Neurons were intracellularlyinjected with trains of somatic current pulses (˜300 PA, lasting ˜4 ms),causing them to fire action potentials at 5 Hz with 100% success rate(FIG. 3A, “I-injection”). Yellow-light pulses were scheduled to occurduring the times when certain spikes (i.e., spikes 7-11 and 17) wouldoccur during the somatic current injection protocol (FIG. 3A). The lightpulses and the somatic current pulses were presented together (FIG. 3A,“I-injection+light”, three trials shown). Spiking was effectivelyblocked during the periods of yellow-light exposure. The rapid onset andoffset kinetics of Halo allowed the deletion of even single spikes. Forinstance, the second yellow-light pulse, timed for silencing just spike17, was able to effectively eliminate spike 17 without affecting thefiring of spikes 16 or 18 at all. The experiment was repeated five timeson each of n=6 neurons (FIG. 3B). During periods when the yellow lightwas off, it was found that somatic current pulses elicited a spike 98.7%of the time. In contrast, during periods when the yellow light was on,somatic current pulses elicited a spike only 1.2% of the time. Thesecond pulse of yellow light reduced the probability of firing spike 17to 3.3%, whereas spikes 16 and 18 still fired 96.7% of the time, notsignificantly different from the spikes at the beginning of the train,before any light exposure at all (X²=1.02, p>0.30). The temporalprecision of Halo in silencing spikes therefore offers the possibilityof creating ultratransient (yet precise and effective) lesions ofactivity in targeted neurons.

A specific embodiment of the present invention includes the use of onemember of the type I opsin family, Channelrhodopsin-2 (ChR2), which hasreceived recent attention for its ability to drive neural excitation inresponse to pulses of blue light (centered around 470 nm). The abilityto drive excitation and inhibition in the same neuron, using twodifferent wavelengths of light, could enable answers to questions forwhich no current technology permits resolution. For example, synchronousneural activity has been correlated with higher-order functions, such asattention and abnormal patterns of neural synchrony that are associatedwith certain neurological and psychiatric disorders. The ability todrive a neuron with balanced but randomly varying excitation andinhibition may allow alteration of the precise timing of membranevoltage fluctuations, in principle permitting neural synchronization ordesynchronization without any side effects, such as alteration of spikerate. This may open up new experiments in testing the causal role ofneural synchrony in behavior and pathology.

Single neurons co-expressing Halo and ChR2, both under control of theCaMKII promoter, were implemented to allow for response torapidly-switched pulses of yellow and blue light with hyperpolarizationsand depolarizations, respectively (FIG. 4A). Poisson trains (λ=100 ms)of rapidly-alternating yellow and blue light pulses elicitedrapidly-alternating hyperpolarizations and depolarizations in the sameneuron (FIG. 4B). In one experiment, the same Poisson train was playedback twice with the first train beginning on a blue pulse (FIG. 4B) andthe second train beginning on a yellow pulse, (FIG. 4C) so that in thesecond trace, depolarizations were converted into hyperpolarizations andvice versa. In principle, these traces should be quite similar, but withinverted voltage scale. Indeed, FIG. 4C shows an inverted tracesuperimposed over the trace in FIG. 4B. The degree of superpositionsuggests that this approach may indeed be a viable method forhigh-fidelity, bi-directional control of neural activity at themillisecond timescale (FIG. 4D).

The inhibition provided by Halo is strong enough to silence neuronsfiring spikes in response to significant intracellular somatic currentinjections (FIG. 3), yet the photocurrents can appear and disappearwithin 10-15 milliseconds of light onset and offset, respectively (FIG.1). Furthermore, the amplitude and timing of responses is reliable fromtrial to trial, and the amplitude of the voltage changes induced bypulses of yellow light does not detectably run down over time (FIG. 2).The use of Halo can be particularly useful for a number of reasons. Forexample, the timescale of inducing of and subsequent release of voltageinhibition by Halo is relatively fast.

According to another embodiment of the present invention, Halo is usedwithout ChR2. Millisecond pulses of light can be used withHalo-expressing cells to induce hyperpolarizations of severalmillivolts, and therefore, may be useful for simulating background orwell-timed synaptic activity. Studying the function of not only specificcell types, but specific classes of inhibitory synapse, can beaccomplished by creating fusion proteins in which Halo is targeted tospecific locations where inhibitory synapses uniquely cluster, such asthe axon initial segment.

The ability to functionally lesion brain regions or cell types in arapidly reversible fashion opens up a large class of experiments inwhich specific neuron populations must be inactivated for precise,sub-second durations during a task. ChR2, another type I opsin whichobligately requires all-trans-retinal for its function, has been shownto function in slices of mammalian brain tissue, or even in the centralnervous system in vivo, without needing any chemical supplementation.Therefore, it is believed that no supplementation will be needed forHalo in the intact mammalian brain and in brain slice experiments. Otherlabs working on classical neural model organisms such as Drosophila andC. elegans have devised ways of delivering all-trans-retinal to thenervous systems of such animals in order to enable ChR2 function, andthus, it is likely that these retinal-delivery protocols would also workfor enabling Halo function in these invertebrates.

The ability to study the causal role of neural synchrony in behavior,neural computation, and neural pathology may be a particularlysignificant role for ChR2 and Halo, working in concert. Thenewly-enabled power to drive both excitation and inhibition ofgenetically-targeted neurons with blue and yellow light seems to beparticularly valuable for probing synchrony by utilizing multiplewavelengths to perform both excitation and inhibition in the samespecimen. The ability to synchronize and desynchronize neurons bybalanced, yet random, patterns of excitation and inhibition may open upnew horizons into understanding the causal role of neural synchrony inbrain function and disease, an area of longstanding, yet growing,interest.

Optical methods for altering neural circuit function have appeal in partbecause in principle they can use technology developed for brainimaging. The ability to use optical fibers to image deep neuralcircuits, for example, also enables the stimulation of deep brainstructures. Two-photon excitation methods may prove valuable for drivingopsin activities, up to 1 mm deep. Another key aspect of optical methodsof neural control is the speed with which activation and inactivationcan take place, since it is trivial to modulate light intensity at highspeeds, faster than most physiologically relevant processes.Nevertheless, non-optical and chemical approaches will continue to findmany powerful uses for reliable, enduring inhibition of specific braincircuits and cell types, especially when large regions of deep braintissue are involved.

From a neuroengineering standpoint, optical prosthetics capable ofinhibiting neural activity may present less-invasive strategies fortreating disorders of neural hyperactivity. ChR2 has already proven tobe well-tolerated in intact mammalian neural circuits for up to a year.If Halo gains a similar track record, it is possible that Halo-enabledprosthetics may open up new horizons in controlling disorders ofexcitable cells, such as epilepsy, depression, neuropathic pain, andcardiac hyperexcitability. In the immediate future, the ability to studythe effects of well-timed neuron or circuit inactivation in animalmodels of disease will rapidly reveal new principles for selectingneural circuit targets for treatment of specific disorders. There arealso implications of the use of Halo in biotechnological scenarios, suchas high-throughput drug screening. Several proposals (and evencommercially-available systems) exist for using electrical stimulationto activate excitable cells, thus facilitating the screening ofdepolarization-gated ion channels. The discovery of drugs that targethyperpolarization-activated channels, such as the family of channelsmediating the hyperpolarization-activated cation currents I(h) and I(f),may be useful for identifying possible drugs for tackling problems suchas absence seizures, bradycardia, and other disorders. An all-opticalmethod for screening for such drugs, which uses light of one frequencyto drive inhibition, and light of another frequency to observe changesin fluorescence of an ion-sensitive chemical or genetically encodedsensor, may revolutionize this process. Thus, Halo not only presents anumber of unique features that enable effective, and rapidly inducibleand reversible, inhibition to be applied to a number of neural circuitquestions, but may open up new horizons in biotechnology as well.

An experimental hippocampal neuron culture, transfection, and survivalassay was implemented according to the following methods. Hippocampalregions CA3-CAI of postnatal day 0 or day 1 Sprague-Dawley rats (CharlesRiver) were isolated and treated with trypsin (1 mglml) for 12 minutes.Digestion was stopped by Hanks solution supplemented with 20% fetalbovine serum and trypsin inhibitor. Tissue was dissociated withsilicone-coated Pasteur pipettes and centrifuged at 1000 rpm at 4° C.for 10 minutes. Dissociated neurons were plated on glass coverslipspre-coated with Matrigel (BD Biosciences) at a rough density ofapproximately two hippocampi per 24 coverslips. Neurons were transfectedusing a commercially available calcium phosphate transfection kit(Invitrogen), at 3-5 days in vitro. GFP fluorescence was used toidentify successfully-transfected neurons, indicating a net transfectionefficiency of ˜7%. All images and electrophysiological recordings weremade on 9-15 day-in-vitro neurons (approximately 4-10 days aftertransfection). Confocal images of transfected neurons were taken with aZeiss LSM 510 confocal microscope. Cell death count was carried out onliving cultures, seven days after transfection, by adding 4 μM ethidiumhomodimer-1 (Invitrogen) to the culture medium for 10 minutes at 37° C.,then washing the cells with Tyrode's solution (see below). GFP-positiveand negative neurons were counted for positive and negative ethidiumfluorescence, in five regions on each of three coverslips for thisviability assay.

An experiment regarding electrophysiology and optical methods wasimplemented according to the following methods. Whole cell patch clamprecording was made on 9-15 day-in-vitro neurons using a Multiclamp 700Bamplifier, connected to a Digidata 1440 digitizer (Molecular Devices)attached to a PC running pClamp 10. During recording, neurons werebathed in Tyrode's solution containing (in mM) 138 NaCl, 2.4 KCl, 2CaCl, 2 MgCl, 10 HEPES, 10 Glucose, 24 sucrose, 10 μM NBQX, 10 μMgabazine and 50 μM D-APV. Borosilicate glass (Warner) pipettes werefilled with a solution containing (in mM) 130 K-Gluconate, 7 KCl, 2NaCl, 1 MgCl2, 0.4 EGTA, 10 HEPES, 2 ATP-Mg, 0.3 GTP-Tris and 20sucrose. Pipette resistance was ˜6 M′Ω, and the access resistance was10-25 M′Ω, which was monitored throughout the voltage-clamp recording.Resting membrane potential was 52-70 mV in current-clamp recording.

Photocurrents were first measured with pairs of 1-second long lightpulses, separated by periods of darkness lasting 1 second, while holdingneurons in voltage clamp at −70 mV, −30 mV and +10 mV to assay theproperties of Halo. Light-induced membrane hyperpolarizations wereinduced by 1 second duration light pulses, separated by periods of 1second darkness, in neurons current-clamped at resting membranepotential. Light pulse trains were synthesized by custom softwarewritten in MATLAB (Mathworks), and then played to the DG-4 light sourcethrough a digital-to-analog converter on the Digidata 1440. For thespike-blockade experiment, spikes were first induced via somatic currentinjection through the patch pipette. Most of the neurons patched easilyfired action potentials with 100% probability, in response to ˜300 pAcurrent injections (4 ms duration). For each neuron, injected somaticcurrent magnitudes guaranteed 100% firing rate of 20 spikes, at a rateof 5 Hz.

A DG-4 optical switch with 300-W xenon lamp (Sutter Instruments) wasused to deliver all light pulses, for Halo or ChR2 activation. A TexasRed filter set (Chroma, excitation 560/55, diachronic 595LP, emission645/75) was used to deliver yellow light to activate Halo. The samediachronic mirror was also used to deliver blue light, but with anexcitation filter 480/40 in the DG-4, to allow ChR2 excitation. Notethat the DC595LP dichroic mirror only reflects 35% of incident 460-500nm light through the objective; custom-coated dichroics that reflectlight all the way into the ultraviolet (as are available from companiessuch as Chroma) would be optimal.

According to one embodiment of the present invention, the survivalreplication, differentiation, or death of cells is modulated byelectrical activity from Halo. With appropriate light pulses,Halo-expressing cells can be guided down any one of these pathways,depending on the precise pattern of stimulation used to drive activationof Halo. A specific electrical activity pattern results in a specificpattern of downstream signal transduction and in a specific cellularfate response. Therefore, targeting Halo to specific cells, thenexposing them to particular light patterns, enables them to be opticallydriven towards survival, differentiation, replication, or death. Thishas many potential applications.

For example, in the case where the target cell is a stem cell,particular patterns of activity will drive the replication ordifferentiation of stem cells (including human embryonic stem cells), ordrive the death of the stem cells (in the case where excessivereplication is desired to cease). If the target cells are tumor orcancer cells, then targeting Halo to those cells will permit the use ofspecific and appropriate patterns of light to drive activity, and thuskill the tumor or cancer cells. If the target cells are immune cells,then silencing the cells can prevent the calcium waves that insure cellsurvival, and reduce the prevalence of autoimmune disease.

Other target cells of this kind may include secretory or organ cells ortheir precursors, cardiac or other muscle cells, or glial cells in thebrain. In each of these cases, it is desirable to control thereplication, differentiation, and death of these cells precisely. Halowill be useful for controlling these things in vitro, in vivo inexperimental animals, or in vivo in humans (before or aftertransplantation into the body)—wherever light can be delivered, such asthrough the skin, via small LEDs, or lasers, or through optical fibersor thin optical endoscopes.

Screening for drugs that modulate ion channel function (e.g., blockingor facilitating ion channel function) can be accomplished using Halo toscreen for drugs that modulate ion channel function. One embodimentinvolves one or more of the following steps:

-   1) stably express Halo in a cell line;-   2) stably express an ion channel of interest (“channel n”) in the    same cell line;-   3) label the cells with a voltage sensitive dye (or other indicator,    see below);-   4) expose said cells to light, and record the fluorescence of the    voltage sensitive dye;-   5) expose said cells to a candidate compound that monitors the    function of channel n; and-   6) expose said cells to light a second time, and record the    fluorescence of the voltage sensitive dye.

If the fluorescence is greater during step 6) than step 4), then thecandidate drug facilitates channel function. If the fluorescence issmaller during step 6) than step 4), then the candidate drug diminisheschannel function. If the fluorescence is equal in steps 4) and 6)(allowing for any bleaching of the dye), then the drug does not affectchannel function. In this way, drugs that affect channel function can bedetected extremely rapidly.

Steps 1) and 2) of the above process may take several hours or days, butthe resulting cell line then suffices for the screening of many (perhapsmillions of) drugs, which modulate channel n. Steps 3), 4), 5), and 6)take only a few seconds each; preferably, steps 4), 5), and 6) each takeless than 1 second. Steps 4), 5), and 6) take place in a robotic devicethat moves a 96- or 384-well plate into the focus of an optical beam(see the last section for details on devices). The wells of the platewould all contain the same cell line, in order to facilitate thescreening of drugs that affect a particular channel, or each well wouldcontain cells of a different cell line, in order to facilitate thescreening of one drug against many different channels (“screeningagainst side effects,” see below).

Step 3 can include the use of a voltage-sensitive dye for fast kinetics;however, another dye (e.g., a calcium-sensitive dye in the case thatchannel n is a calcium channel) could also serve to indicate whetherchannel function is modulated by the drug. Genetically encodedindicators of voltage or calcium would also be useful for reading outthe activity of the cell (e.g., FLASH, GCaMP2, cameleon, etc.). In thiscase, these indicators would be stably expressed in the cell line aswell. Other methods of reading out whether the drug had an effect couldalso be useful for supplementing this readout (e.g., immunostaining forthe phosphorylation of a site that is phosphorylated during or afterperiods of ion channel activity).

Blindness and other sensory deficits affect millions of peopleworldwide, severely impacting their quality of life. Halo can betargeted to somatic cells in the human patient to provide a type ofsensory prostheses. For example, some forms of blindness destroyphotosensor function but leave signal processing in downstream neuronsintact. In such diseases, such as macular degeneration or retinitispigmentosa, targeting Halo to the “off” retinal ganglion cells (e.g., byinjecting viruses expressing Halo into the retinal cell layers insidethe eye) would enable restoration of visual function. As light increasesin the environment, Halo would inhibit the “off” cells, causingincreased visual responses in the brain. In such patients treated withHalo targeted to retinal ganglion cells, the retinal ganglion cellswould themselves become photosensitive, enabling vision with resolutioncomparable to the native eye, and not requiring invasive technologybeyond that point. Halo is sufficiently sensitive to detect sunlight(power ˜1 kW/m{circumflex over ( )}2), with maximal sensitivity in thepart of the spectrum that is greatest in sunlight. Expressing Halo in aretinal cell, accompanied with a projection device that would amplifythe ambient light, would enable vision inside or in lowlight conditions.

Another implementation of Halo involves situations where the centralnervous system neurons in a person are infected with virus expressingHalo (or otherwise come to express Halo). These neurons would then beinhabitable by pulses of yellow light. This gene therapy approach wouldtherefore allow optical inhibition of precise neuronal targets in thebrain. If the targeted neurons are epileptic, this would enablesilencing of those cells without needing ablative surgery. If thetargeted neurons were in the frontal cortex or other parts of the brain,these light-sensitive neurons would permit optical modulation of emotionor cognition. If the targeted neurons were in the spinal cord, neuronsthat mediate pain stimuli could then be inhibited by light.

In general, such a gene therapy approach opens up a new kind ofgeneralized prosthetic in defined parts of the nervous system. Theprosthetic allows light to be converted into neural activity.

In another instance, Halo is targeted to specific and different parts ofa cell. For example, targeting Halo to the axon hillock using the AIS(axon initial segment) targeting sequence allows more powerfulinhibition. Fusing Halo to a targeting sequence of DNA, so that theresultant protein contains both Halo and the targeting peptide, allowsHalo to be sent to the presynaptic terminal, the postsynaptic terminal,the nucleus, or other intracellular compartments. Such targetingsequences include PDZ domains, glutamate and GABA receptor C-terminalsequences, ion channel C-terminal sequences, presynaptic scaffoldingtargeting sequences, and other targeting sequences. These versions ofHalo can then be used to trigger specific intracellular signalingevents, including those important for neuroprotection, memory, or otherenduring signaling functions.

In a combinatorial fashion, these reagents could complement the otherapplications of Halo. For example, these reagents could be useful fordrug screening (e.g., finding drugs that modulate the function of achannel in a particular subcellular compartment). These reagents couldalso be useful for prosthetic devices (e.g., driving activity on thedendrites of a neuron, to more closely mimic natural synaptic activity).

Various embodiments, including but not limited to those involving drugscreening, employ an optical imaging device containing 1) a light source(LED, lamp, laser) for illuminating the cell expressing Halo and drivinga change in cell voltage, 2) a light source for illuminating a dye orindicator, possibly the same light source as used for driving thevoltage change, and 3) a switch for alternating between the two lightsources or a beamsplitter for simultaneous non-interfering delivery ofboth kinds of light. The fluorescence of the dye or indicator would bemeasured by a sensor (CCD camera, PMT, or photodiode). This kind ofdevice can be useful for ion channel drug screening, as described above.The device itself consists of a robotic arm for moving a plate (e.g., a384-well plate) through the arena where the light sources and sensor arepresent.

In one embodiment, diagnostic applications, as mentioned herein, use acombined light source imaging device. For example, taking cells from apatient, expressing Halo in them, and then exposing them to light, canbe used to reveal patient-specific ion channel syndromes in biopsysamples or in cells of the circulatory system.

For various implementations, an implantable or head-mounted LED, orother small light source can be used. Such a light source can beimplanted under the skin, under the skull, deep within the brain, ordeep within another organ of interest, in which Halo-expressing cellsare also located (either exogenously introduced, or endogenously locatedand targeted with a virus). This device can be used for stimulating Haloin cells located directly adjacent to the light source. A strip of LEDs,each individually controllable, is useful. For the example of thecortical implant, a 2-dimensional array of LEDs is useful.

For medical applications, various embodiments have LEDs that areremotely powered. A remotely-powered LED can be made, for example, bycombining an LED in a closed-loop series circuit with an inductor. Thiswould allow radiofrequency (RF) energy or rapidly changing magneticfields (e.g., delivered by a transcranial magnetic resonance (TMS) coil)to temporarily power-up the inductor, and thus the connected LED,allowing local delivery of light, even deep in a brain structure. Incertain embodiments, such a device is implanted under the skin, underthe skull, deep within the brain, or deep within another organ ofinterest in which Halo-expressing cells are also located (eitherexogenously introduced, or endogenously located and targeted with avirus). Optionally, another device is used to remotely deliver RF ormagnetic energy (e.g., placed nearby or worn on the patient) foractivating the implanted device.

N. pharaonis halorhodopsin with mammalian-optimized codon usage wassynthesized as a DNA sequence according to the sequence listing providedon the following page as Sequence Listing A.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the invention.Based on the above discussion and illustrations, those skilled in theart will readily recognize that various modifications and changes may bemade to the present invention without strictly following the exemplaryembodiments and applications illustrated and described herein. Forinstance, such changes may include the use of digital logic ormicroprocessors to control the emitted light. Such modifications andchanges do not depart from the true spirit and scope of the presentinvention, which is set forth in the following claims.

SEQUENCE LISTING A

The N. pharaonis halorhodpsin with mammalian-optimized codon usage wassynthesized according to the following DNA sequence (876 base pairs).

ATGACTGAGACCCTCCCACCCGTGACTGAAAGCGCCGTCGCTCTGCAAGCAGAGGTTACCCAGCGGGAGCTGTTCGAGTTCGTCCTCAACGACCCCCTCCTGGCTTCTAGCCTCTACATCAACATTGCTCTGGCAGGCCTGTCTATACTGCTGTTCGTCTTCATGACCAGGGGACTCGATGACCCTAGGGCTAAACTGATTGCAGTGAGCACAATTCTGGTTCCCGTGGTCTCTATCGCTTCCTACACTGGGCTGGCATCTGGTCTCACAATCAGTGTCCTGGAAATGCCAGCTGGCCACTTTGCCGAAGGGAGTTCTGTCATGCTGGGAGGCGAAGAGGTCGATGGGGTTGTCACAATGTGGGGTCGCTACCTCACCTGGGCTCTCAGTACCCCCATGATCCTGCTGGCACTCGGACTCCTGGCCGGAAGTAACGCCACCAAACTCTTCACTGCTATTACATTCGATATCGCCATGTGCGTGACCGGGCTCGCAGCTGCCCTCACCACCAGCAGCCATCTGATGAGATGGTTTTGGTATGCCATCTCTTGTGCCTGCTTTCTGGTGGTGCTGTATATCCTGCTGGTGGAGTGGGCTCAGGATGCCAAGGCTGCAGGGACAGCCGACATGTTTAATACACTGAAGCTGCTCACTGTGGTGATGTGGCTGGGTTACCCTATCGTTTGGGCACTCGGCGTGGAGGGAATCGCAGTTCTGCCTGTTGGTGTGACAAGCTGGGGCTACTCCTTCCTGGACATTGTGGCCAAGTATATTTTTGCCTTTCTGCTGCTGAATTATCTGACTTCCAATGAGTCCGTGGTGTCCGGCTCCATACTGGACGTGCCATCCGCCAGCGGCACACCTGCCGATGACTGA).

The Halo-GFP fusion protein was generated by PCR amplification of theHalo gene with primers 5′ GAATTCGCCACCATGACTGAGACCCTCCCACCCGTG and3′GGATCCGTCATCGGCAGGTGTGCCGCTGGC and inserted into the EcoRI and BamHIcites of pEGFP-N3 (Clontech), which has the CMV promoter. The Halo-GFPfusion protein sequence was then PCR amplified with primers5′CCGGTGCCACCATGACTGAGACCCTCCCACCCGTG and3′GAATTCTTACTTGTACAGCTCGTCCATCGG and inserted into lentiviral vectorFCK(1.3)GW containing the CaMKII promoter via Agel and EcoRI sites. Allconstructs were verified by sequencing. The channelrhodopsin constructused in various experiments, FCK-hCmC, contains the human/mammaliancodon-optimized gene ChR2 fused to fluorescent protein mCherry, underthe CaMKII promoter.

What is claimed is:
 1. A method for screening a candidate agent thatmodulates ion channel function in a mammalian cell, the methodcomprising: (i) expressing in a mammalian cell a light-driven chlorideion pump from Natronobacterium pharaonis (NpHR); (ii) expressing in themammalian cell an ion channel; (iii) labeling the mammalian cell with avoltage sensitive dye; (iv) exposing the mammalian cell to a firstlight; (v) recording a first fluorescence signal of the voltagesensitive dye; (vi) exposing the mammalian cell expressing NpHR to thecandidate agent; (vii) exposing the mammalian cell expressing NpHR to asecond light; and (viii) recording the second fluorescence signal of thevoltage sensitive dye, and wherein if the second fluorescence signal ofthe voltage sensitive dye is greater than the first fluorescence signalof the voltage sensitive dye, then the candidate agent facilitates ionchannel function.
 2. The method of claim 1, wherein if the firstfluorescence signal of the voltage sensitive dye is equal to the secondfluorescence signal of the voltage sensitive dye, the candidate agentdoes not have an affect ion channel function.
 3. The method of claim 1,wherein if the second fluorescence signal of the voltage sensitive dyeis smaller than the first fluorescence signal of the voltage sensitivedye, than the candidate agent diminishes ion channel function.
 4. Themethod of claim 1, wherein the ion channel is a Channelrhodopsin-2(ChR2) ion channel.
 5. The method of claim 4, wherein the first light isa yellow light or a blue light, wherein the yellow light activates theNpHR ion pump expressed in the mammalian cell and the blue lightactivates the ChR2 ion channel expressed in the mammalian cell.
 6. Themethod of claim 1, wherein the voltage sensitive dye is acalcium-sensitive dye.
 7. The method of claim 1, wherein the secondlight is a yellow light or a blue light, wherein the yellow lightactivates the NpHR ion pump expressed in the mammalian cell and the bluelight activates the ChR2 ion channel expressed in the mammalian cell.